Realistic in vitro models are critical in the drug development process. In this study, a novel in vitro platform is employed to assess drug penetration and metabolism. This platform, which utilizes a 3D printed fluidic device, allows for dynamic dosing of three dimensional cell cultures, also known as spheroids. The penetration of the chemotherapeutic irinotecan into HCT 116 colon cancer spheroids was examined with matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). The active metabolite of irinotecan, SN-38, was also detected. After twenty-four hours of treatment, SN-38 was concentrated to the outside of the spheroid, a region of actively dividing cells. The irinotecan prodrug localization contrasted with SN-38 and was concentrated to the necrotic core of the spheroids, a region containing mostly dead and dying cells. These results demonstrate that this unique in vitro platform is an effective means to assess drug penetration and metabolism in three dimensional cell cultures. This innovative system can have a transformative impact on the preclinical evaluation of drug candidates due to its cost effectiveness and high throughput.
In vitro models for screening new cancer chemotherapeutics often rely on two-dimensional cultures to predict therapeutic potential. Unfortunately, the predictive power of these models is limited, as they fail to recapitulate the complex three-dimensional environments in tumors that promote the development of a chemoresistant phenotype. In this study, we describe the preparation and characterization of paper-based cultures (PBC) engineered to assess chemotherapeutic effectiveness in three dimensional, diffusion-limited environments. Similar environments are found within poorly vascularized tumors. These cultures, which are assembled by stacking together cell-laden paper scaffolds to yield thick tissue-like structures, generate monotonic gradients vertically through the culture and provide distinct chemical environments for each scaffold. After prolonged incubation, the scaffolds can simply be peeled apart and analyzed to relate cellular responses to the chemical environment experienced at that scaffold. Through fluorescence imaging, viable and proliferative cell populations were mapped within a stacked culture and found to be most abundant in scaffolds close to the nutrient-rich medium. By adjusting the cell density, we modulated the spatiotemporal evolution of oxygen gradients across the cultures and correlated these environmental changes with cellular sensitivity to SN-38 exposure. From these results, we showed that differences in the oxygen gradients produced cellular populations with significantly different chemosensitivities. Through this work, we highlight PBCs ability to serve as an analytical model capable of determining chemotherapeutic effectiveness under a range of chemical environments.
For a patient with metastatic colorectal cancer there are limited clinical options aside from chemotherapy. Unfortunately, the development of new chemotherapeutics is a long and costly process. New methods are needed to identify promising drug candidates earlier in the drug development process. Most chemotherapies are administered to patients in combinations. Here, an in vitro platform is used to assess the penetration and metabolism of combination chemotherapies in three-dimensional colon cancer cell cultures, or spheroids. Colon carcinoma HCT 116 cells were cultured and grown into three-dimensional cell culture spheroids. These spheroids were then dosed with a common combination chemotherapy, FOLFIRI (folinic acid, 5-fluorouracil, and irinotecan) in a 3D printed fluidic device. This fluidic device allows for the dynamic treatment of spheroids across a semipermeable membrane. Following dosing, the spheroids were harvested for quantitative proteomic profiling to examine the effects of the combination chemotherapy on the colon cancer cells. Spheroids were also imaged to assess the spatial distribution of administered chemotherapeutics and metabolites with MALDI-imaging mass spectrometry. Following treatment, we observed penetration of folinic acid to the core of spheroids and metabolism of the drug in the outer proliferating region of the spheroid. Proteomic changes identified included an enrichment of several cancer-associated pathways. This innovative dosing device, along with the proteomic evaluation with iTRAQ-MS/MS, provides a robust platform that could have a transformative impact on the preclinical evaluation of drug candidates. This system is a high-throughput and cost-effective approach to examine novel drugs and drug combinations prior to animal testing.
Drug resistance is a prevalent phenomenon that decreases the efficacy of cancer treatments and contributes to cancer progression and metastasis. Weakening drug resistant cancer cells prior to chemotherapy is a potential strategy to combat chemoresistance. One approach to damage resistant cancer cells is modulation of nutritional intake. The combination of nutrient restriction with targeted compound treatment results in pronounced molecular changes. This study provides valuable information about augmenting existing chemotherapeutic regimes with simultaneous glucose restriction and autophagy inhibition in colorectal cancer cells. In this study, we explore the chemical pathways that drive the cellular response to nutrient restriction, autophagy inhibition and the chemotherapy irinotecan using global quantitative proteomics and imaging mass spectrometry. We determined that significant pathways were altered including autophagy and metabolism via glycolysis, gluconeogenesis, and sucrose degradation. We also found that period circadian clock 2 (PER2), a tumor suppressor protein, was significantly up regulated only when glucose was restricted with autophagy inhibition and chemotherapy. The upstream regulators of these differentially regulated pathways were determined to have implications in cancer, showing an increase of tumor suppressor proteins and a decrease in nuclear protein 1 (NUPR1) an important protein in chemoresistance. We also evaluated the phenotypic response of these cells and discovered autophagy inhibition and chemotherapy treatment increased apoptosis and decreased cell clonogenicity and viability. When glucose restriction was combined with autophagy inhibition and chemotherapy, all of the phenotypic results were intensified. In sum, our results indicate that glucose metabolism is of great importance in the ability of cancer cells to survive chemotherapy. By weakening cancer cells with glucose restriction and autophagy inhibition prior to chemotherapy, cancer cells become more sensitive to therapy.
Many potential chemotherapeutics fail to reach patients. One of the key reasons is that compounds are tested during the drug discovery stage in two-dimensional (2D) cell cultures, which are often unable to accurately model in vivo outcomes. Three-dimensional (3D) in vitro tumor models are more predictive of chemotherapeutic effectiveness than 2D cultures, and thus, their implementation during the drug screening stage has the potential to more accurately evaluate compounds earlier, saving both time and money. Paper-based cultures (PBCs) are an emerging 3D culture platform in which cells suspended in Matrigel are seeded into paper scaffolds and cultured to generate a tissue-like environment. In this study, we demonstrate the potential of matrix-assisted laser desorption/ionization–mass spectrometry imaging with PBCs (MALDI-MSI-PBC) as a drug screening platform. This method discriminated regions of the PBCs with and without cells and/or drugs, indicating that coupling PBCs with MALDI-MSI has the potential to develop rapid, large-scale, and parallel mass spectrometric drug screens.
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